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Two monoclonal antibodies (mAb), MEC 7.46 (IgG1) and MEC 13.3 (IgG2a) that specifically recognize mouse endothelial cells (EC) of blood vessels, were produced immunizing a Lewis rat with a polyoma middle T transformed EC line. Antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and by immunofluorescence on different cultured cell lines and by immunoperoxidase staining on frozen sections of various mouse normal and inflammatory tissues. Both mAbs reacted with eight transformed endothelial lines tested in vitro, but were consistently negative on various cell lines of different histological origin. Reactivity was not altered by preexposure of the cell lines to IL-1. Microscopic immunofluorescence analysis showed that the MEC mAbs localized at the cell-cell contacts in EC. Immunohistochemical staining of various mouse tissue was always restricted to the EC of all blood vessels of the organ considered. Staining of the endothelial lining of blood vessels was greater at cell-to-cell contacts. Weak reactivity was detected in bone marrow and spleen megakaryocytes. This picture was not altered in inflamed and tumor tissues. In the developing mouse embryo, MEC 13.3 specifically stained proliferating and sprouting endothelium in all organs and tissues examined. Both MEC 7.46 and MEC 13.3 mAbs were able to precipitate a molecule with an apparent molecular mass of 130 kDa from endothelioma lysates. The protein was synthesized by the cells and exposed on the cell surface. Immunodepletion analysis indicated that MEC 13.3 recognized a molecule related to the murine from of PECAM or CD31. We believe that these mAbs are promising tools for the identification of murine EC and for studying their ontogenesis and functions.